Citrus reticulata Blanco, F. Rutaceae) against sarcoptic mange-in-rabbits. Analysis of the oil's GC–MS identified a total of 20 compounds, accounting for 98.91% of all compounds found. Mandarin peel oil topical application improved all signs of infection, causing a scabicidal effect three days later, whereas in vitro application caused complete mite mortality one day later. In comparison to ivermectin, histopathological analysis showed that the epidermis' inflammatory-infiltration/hyperkeratosis-had disappeared. In addition to TIMP-1, the results of the mRNA gene expression analysis showed upregulation of I-CAM-1-and-KGF and downregulation of ILs-1, 6, 10, VEGF, MMP-9, and MCP-1. The scabies network was constructed and subjected to a comprehensive bioinformatic evaluation. TNF-, IL-1B, and IL-6, the top three hub protein-coding genes, have been identified as key therapeutic targets for scabies. From molecular docking data, compounds 15 and 16 acquired sufficient affinity towards the three screened proteins, particularly both possessing higher affinity towards the IL-6 receptor. Interestingly, it achieved a higher binding energy score than the ligand of the docked protein rather than displaying proper binding interactions like those of the ligand. Meanwhile, geraniol (15) showed the highest affinity towards the GST protein, suggesting its contribution to the acaricidal effect of the extract. The subsequent, MD simulations revealed that geraniol can achieve stable binding inside the binding site of both GST and IL-6. Our findings collectively revealed the scabicidal ability of mandarin peel extract for the first time, paving the way for an efficient, economical, and environmentally friendly herbal alternative for treating rabbits with Sarcoptes mange.The current study investigated the scabicidal potential of Egyptian mandarin peel oil ( Sarcoptes scabiei) is a serious infectious disease that invades humans and animals all over the world1. The mites are highly adapted to contact with their host as contagious, burrowing, and obligate parasites. Sarcoptic mange Grower pig production is negatively impacted by adult female mites; because they mate on the skin's surface, burrow into the skin, lay eggs, and cause irritations that can lead to bleeding, reduced feeding and development, chronic stress, and decreased welfare3. The clinical picture represents chronic hyperkeratotic, which is characterized by the presence of aural crusts and many mites on the animal4. Similar to people, rabbits are susceptible to Sarcoptes infection, or mange, which reduces production and causes economic losses for rabbits, especially in the absence of effective treatment5. Therapy options include the systemic treatment of macrocyclic lactones, local administration of amitraz or pyrethroids, or both7. Despite their long history of effectiveness in treating mange, their extensive use has led to a decline in effectiveness because of the emergence of drug resistance. Thus, it is crucial to create novel scabicides that are both efficient and secure in order to treat and control mammalian scabies6.Sarcoptic mange , C. nobilis Loureiro , C. deliciosa Tenore , and C. reticulata Blanco (common mandarins)17. Mandarins are one of the main Citrus fruits grown in many countries such as China, Brazil, USA, India, Mexico, Spain, etc. The fruits have a great commercial worth for their essential oils and other fragrant compounds, even though they are primarily used to make pastries18. A lot of beverages, candies, cookies, and desserts use Citrus flavours19, while the peels of C. reticulata are used to flavour alcohol19. Citrus reticulata EO shown an anti-proliferative activity against rat pulmonary fibrosis produced by bleomycin (BLM) and protective properties against human embryonic lung fibroblasts (HELFs). The method is believed to involve correcting the imbalance between oxidation and antioxidation, lowering collagen deposition and fibrosis, and down-regulating lung tissue expressions of connective tissue growth factor (CTGF) and mRNA20. Due to its high d-limonene concentration21, C. reticulata EO demonstrated a moderate level of radical scavenging action22. Mandarin oil is well known for its broad spectrum antibacterial and antifungal actions. It inhibits the growth of several bacteria including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus23, as well as several fungi including Penicillium italicum, P. chrysogenum, P. digitatum, Aspergillus niger,-A. flavus, Alternaria alternata, Curvularia lunata, Rhizoctonia solani, Fusarium oxysporum, and-Helminthosporium oryzae26.Some of the most coveted ,-in vivo,-histopathology,-mRNA-expression, and network/in silico analysis, the extract's scabicidal potential against-Sarcoptic-mange-in-rabbits has been investigated, allowing for the incorporation of natural candidates to proper and secure management of infectious diseases. The present investigation's framework is shown in Fig. The GC–MS profiling of mandarin peel oil has been used in the current study. Additionally, for the first time, through-in vitro27.Plant materials and experiments were conducted in accordance with relevant institutional, national, and international guidelines. The study took place according to the ethical committee's permission number of 9/5/2022 at Deraya College. It was done in accordance with the National Institute of Health's guidelines for the care and use of laboratory animals and ARRIVE guidelinesC. reticulata cultivated fruits were harvested from a house garden on Atia Street in Beni-Suef, Egypt. A voucher specimen (2021-BuPD-88) was deposited at Pharmacognosy-Department, Faculty-of-Pharmacy, Beni-Suef-University, Egypt.In January 2021, 29.Using the Clevenger apparatus, the fresh peels (0.5 kg) were hydrodistillated for two hours at 75 °C. The oil was gathered, dried over anhydrous sodium sulphate, and kept in airtight amber glass vials at 4 °C for storage. On the basis of the plant material's fresh weight, the yield (v/w%) was computed30. The GC–MS apparatus combines a thermal mass spectrometer detector (ISQ single quadrupole mass spectrometry) with a TRACE GC ultra-high performance gas chromatograph . A TR-5 MS column  was installed in the GC–MS system. For the analyses, He-lium was used as the carrier gas, and the split ratio was set at 1:10 using the following temperature program: 60 C for 1 min, followed by 4.0 C/min to 240 C and a 1-min hold. At 210 °C, the injector and detector were maintained. One-liter samples of the mixes were always administered as diluted samples . By using a spectral range of m/z 40–450 and electron ionization (EI) at 70 eV, mass spectra were produced. Using AMDIS software (www.amdis.net), the chemical components of the essential oil were deconvoluted and identified by their retention indices , mass spectra matching to genuine standards, and retention times (when available).  Wiley spectral library collection32.Gas chromatography-mass spectrometry (GC/MS) was used to perform chromatographic analysis on the oil recovered from peels2O2) was used to determine H2O2 scavenging activity that reflects the anti-oxidative capacity of the peel oil. Colorimetric analysis was used to estimate the residual H2O233. In brief, 20 µl of the sample was mixed with 500 µl of H2O2 and incubated at 37 °C for 10 min. 500 l of the enzyme/3, 5-dichloro-2-hydroxyl-benzensulfonate solution were then added, and it was incubated at 37 °C for 5 min. The colored product's intensity was quantified colorimetrically at-510-nm. A positive control was ascorbic acid. By comparing the test results to those of the control group, the percentage-of H2O2-scavenging activity-was calculated and applying the following formula:The reaction with a defined amount of exogenously provided hydrogen peroxide  using Graph pad prism 7 software.IC34. In a Tris-HCL solution  containing 90 l of NBT (0.3 mM), 90 l of NADH (0.936 mM), 0.1 ml of peel oil , and 0.8 ml of Tris-HCl buffer, superoxide anion radicals were produced . After adding 0.1 ml of PMS solution (0.12 mM) to the mixture, the reaction was started. The mixture was then incubated at 25 °C for 5 min, during which time the absorbance was measured at 560 nm. Ascorbic acid was used as a model substance. Using the formula below, the percentage inhibition was calculated by comparing the test results to those of the control:The scavenging activity of superoxide anion was measured50 was estimated by doing the test at four different concentrations and using the GraphPad Prism 7 software.ICAdult-mites-were collected from rabbits that were-naturally-infected, Deraya University, Minia, Egypt's Animal House. Scraped from the borders of the lesions, the infected skin samples were then shifted-to-petri plates and-incubated within a biochemical-oxygen-demand (BOD) for an incubator for 30 min at 35 °C.A petri dish containing mites was filled with 2 ml of diluting extract (20%), along with the plates were then incubated-in-BOD. Reaction observations were made-at 1, 12, and 24 h after application. Petri plates were incubated at an ambient temperature of 25 °C and with a relative moisture of 75%, with a 5% ivermectin (1 cm3/l) group as the positive-control-and distilled water as the-negative-control. By stimulating the mites with a needle, the death of the mite was confirmed; the mite was deemed dead if it showed no response.35. Each group were kept in a separate cage, and each group received treatment by dipping the infected ears once daily. Steel hoppers were used to feed all of the rabbits, and water was available at all times. The rabbits were observed every two days to assess their clinical recovery. The goal was to find any signs of improvement in the lesions, such as the absence of irritation and redness, cutaneous smoothing, the start of the development of hair from the infection, and the cessation of scab development10. Skin scrapings from each rabbit's sick and healed areas were taken every three days, and Throughout the course of the therapy, they were microscopically investigated to check for sarcoptic mites with a LEICA, DM1000 microscope with a digital camera 10.The study took place on male adult rabbits for 4 weeks (weighing 2.8–3.2 kg) that were infected. The animals' ears showed clinical indicators of mange infection, such as hyperkeratinization, inflammation, redness, itching, and irritability. Microscopic mite identification in skin scrapings further corroborated this. Four groups of five rabbits each were made up of twenty animals, as follows: Five rabbits made up the normal group, the paraffin oil-positive-control-group. The-ivermectin-treated-group (5%-ivermectin). The peel-oil-group (20%-peel-oil in paraffin-oil). Paraffin oil, which is a-mineral-oil, was reportedly chosen as a diluent for the peel oil because it has little impact-on-mites36.Tissue samples were collected at zero and three weeks following the start of the course of therapy via 20% peeling oil as well as ivermectin from healthy and infected ears. Following that, samples were dried in ethyl alcohols of increasing strength, sterilized via xylene, infused with paraffin that had been melted at 55–60 °C, and finally inserted into paraffin wax. The samples were then preserved in 10% buffered formalin. Deparaffinized, rehydrated, and stained with hematoxylin and eosin (H & E), “3–5 m thick” tissue sections were examined using-a-light-electron-microscope31. Table Using-a-digital-homogenizer-, 100 mg of the tissues under investigation were homogenised in 1 ml of TRIzolTM RNA Extraction Reagent . RNA extraction from the biopsy sample was done in accordance with the manufacturer's instructions. RevertAid H-minus First Strand cDNA Synthesis Kits  were used to create cDNA from the extracted RNA for comparable amounts of total RNA in all samples. The-qRT-PCR-was carried out on-the-Applied-Biosystems Step One Plus system using the cDNA as a template. The primers were created using the NCBI primer blast software and were produced by Invitrogen. Using the GAPDH gene as a housekeeping gene, data were analyzed using the 2CT approachhttps://www.cytoscape.org/)37 and by lunching STRING disease query tool incorporated in it which retrieves network for the top human proteins associated with the queried disease from a weekly updated web source of diseases database (https://string-db.org/)38 Scabies was chosen as the search term, and "Human sapiens" was chosen as the species type. The confidence score was set to 0.4, and the default settings for the remaining parameters were used to create the PPI network39.Using Cytoscape 3.9.1 software , maximum neighbourhood component (MNC), the density of maximum neighborhood component (DMNC), and maximal clique centrality (MCC), as well as bottleneck, eccentricity, closeness, radiality, betweenness, stress, and clustering coefficient. Cytoscape is regarded as a useful exploration interface for the most significant nodes in PPI networks42.We employed a freely accessible bioinformatics web tool in the current investigation (ShinyGO v0.76.3). Using the many bioinformatics databases accessible, it is possible to perform both gene ontology enrichment analysis and pathway enrichment analysis. ShinyGO was used to perform the gene ontology and enrichment analysis on the 16 genes to determine the cellular elements, molecular functions, and biological processes that were impacted by this set of genes. ShinyGO retrieves comprehensive descriptions of biological signal transduction pathways from numerous databases43. These cy-tokines have been identified as one of the primary molecules responsible for allergic Th2-type inflammation in the immunological response to scabies, along with TNF-, which is significant in alternative macrophage activation44. GSH, which is linked to the scabies defense system, takes part in a variety of processes crucial to the preservation of cells from oxygen and free radical oxidative damage45, Its distinctive anti-oxidant action makes it a potential target for the oil's acaricidal impact46. In our docking investigation, we validated the ligand and visualized the many docked poses using the computer programme MOE 2019.010. TNF- complexed with its ligand (PDB ID code: 2AZ5) is the last one, and GST is the other protein target of the mite delta class. The first protein target is (IL-1), depicted by the protein's PDB ID code of 6Y8M in co-crystallization with IL-6, as reflected by PDB ID code 1ALU, and its inhibiting ligand SX2 (a-bromo-amido-pyridine-derivative)47 represented by proteins (PDB ID code: 3EIN), the selected targets were acquired via the web from the Database of Proteins (http://www.rcsb.org/pdb).The methodologies of molecular docking intend to predict the best binding orientation of a ligand to a receptor. It proposes several suitable poses of the ligand within the active or docking site of a receptor molecule.in this study, twenty compounds that were identified underwent an in silico study by using screening for three different important-protein-targets that are heavily involved in the scabies infection process, as well as screening for potential targets at the mite itself as an acaricidal effect, in an attempt to get deep inside the mechanistic anti-scabietic effect of orange oil. The chosen targets include IL-1, which is highly effective in stimulating T cells with regulatory functions, and IL-6, which is involved in the formation of Th17 lymphocytes and the release of IL-1749. The Charmm-36 force field is implemented in this piece of software. The protein structure was examined for missing hydrogens, the protonation states of the amino acid residues were set (pH = 7.4), and the co-crystallized water molecules were removed using the QwikMD toolkit of the VMD software. The entire assembly was then packed into a 20 solvent buffer containing 0.15 M Na + and Cl- ions in an orthorhombic box of TIP3P water. After 5 ns of equilibration, the systems were subjected to an energy minimization protocol. Force Field Toolkit (ffTK), a plugin for the VMD software, was used to determine ligand properties and topologies. After the parameters and topology files were prepared, they were imported into VMD so that the protein–ligand complexes could be read accurately, and the simulations could be run.The MD simulations were carried out using NAMD 3.0.0. softwareThe data were tabulated using the statistical programme-GraphPad-Prism-version-9 . To evaluate statistical differences between the groups, the ANOVA test was performed, followed by the-Bonferroni-post-hoc-test-for multiple-comparisons. The threshold for statistical significance is a p-value of 0.05 or less.C. reticulata peels gave 2.6% v/w volatile oil fresh weight, being colourless with a characteristic odor, lighter than water, clear, transparent, and not viscous at room temperature as well as at 4 °C. GC–MS analysis was used to identify a total of 20 compounds, accounting for 98.91% of all compounds found  which represented the major oil fraction, to oxygenated cyclic hydrocarbon ((-)-isomenthone 10, terpinen-4-ol 11, (-)-carvone 14, 3.32%), and oxygenated acyclic hydrocarbon , acyclic hydrocarbon , bicyclic hydrocarbon , to oxygenated bicyclic hydrocarbon  -valencene 20, 1.00%), to a tricyclic hydrocarbon  , represents mainly limonene (50.42%), myrecene (3.03%), and α-terpineol (1.19%), while sesquiterpene (12.98%) represents mainly α-copaene (1.49%), β-copaene (1.30%), and α-humulene (1.23%). The Indian C. reticulata oil is characterised by its high content of fatty acids (8.73%) and aldehyde content (7.08%), mainly n-hexadecanoic acid (5.65%) α-sinensal (3.14%)51. The essential oil isolated from fully matured, ripened Indian fruit peels of C. reticulata, on the other hand, contained 37 different components (99%). The primary ingredients included limonene (46.7%), geranial (19.0%), neral (14.5%), geranyl acetate (3.9%), geraniol (3.5%), -caryophyllene (2.6%), nerol (2.3%), neryl acetate (1.1%), and others26.According to the literature, the chemical composition of essential oils varies depending on the age of the plant, harvesting time, geographical location, and environmental conditionsC. reticulata grown in Burundi contained 58 constituents52. The most prevalent chemical category was monoterpene hydrocarbons (94.7%). Limonene accounted for 84.8% of the total composition, with -terpinene (5.4%), myrcene (2.2%), and -pinene (1.1%) following. Germacrene D and valencene were the primary components of the sesquiterpene hydrocarbons, which made up only 0.2% of the total composition. Compounds containing oxygen from different chemical groups made up 2.3%52. The two main chemical groupings were terpene alcohols (0.7%) and aliphatic aldehydes (0.7%). Linalool (0.7%), octanal (0.5%), and decanal (0.2%) made up the bulk of the mixture. In concentrations of 0.1%, octyl acetate, α-sinensal, decanol, and perillaldehyde were present. Thymol, α-sinensal, methyl thymol, as well as the acetate esters bornyl, ɣ-terpinyl, geranyl, citronellyl, and decyl acetates, were all found at concentrations of less than 0.05%52.The essential oil constituents reported in C. reticulata cultivated in Algeria were reported to contain 24 constituents. Monoterpene hydrocarbons accounted for the most abundant chemical group (89.56%). The main components were limonene (67.04%), -terpinene (15.50%), and -pinene (2.75%). Sesquiterpene hydrocarbons accounted for a minor quantity (3.26%), where l-caryophyllene was the main constituent53.The essential oil constituents of C. reticulata cultivated in different regions corroborates some commonalities. Consequently, limonene, a hydrocarbon monoterpene, is invariably the most common ingredient in essential oils made from Citrus peels, making up typically between 60 and 70 percent of the oil. However, limonene can show lower levels, as in fully matured, ripened Indian fruit peels of C. reticulata, in which it can decrease to 46%26. Also prevalent are the following substances: monoterpenes, which typically account for less than 15%, γ-terpinene, myrcene, and α-pinene, which can reach an abundance of 6.0%, 3.6%, and 1.5%, respectively.The literature review on essential oil components in Non-terpenoid or terpenoid compounds  are reported to be present (1–10%) or absent according to the cultivated region, but there are no commonalities among studies reporting these compounds to have an impact on the essential oil activity or not. Sesquiterpene hydrocarbons are the most varied group of all known chemicals, and this is true for the majority of species. The most prevalent groupings also frequently include oxygenated monoterpene alcohols and monoterpene hydrocarbons.2O2. The outcomes showed that mandarin peel oil had H2O2 scavenging capacity at a concentration of 1000 µg/mL increased in an exceedingly dose-dependent manner, compared with a standard-ascorbic-acid (IC50 = 139.2 µg/mL). This means that the higher the concentration of the oil, the more effectively it scavenges the H2O2 radicals  achieved a remarkable acaricidal impact. The mites displayed a slow movement that began at one-hour post-application-(PA) and terminated at 24 PA via 99 percent death rates, as determined by microscopic analysis.Sarcoptes scabiei. These animals suffered from itching, congestion, scratching, and anorexia, while those treated with mandarin peel oil (20% peel oil in paraffin oil) showed a gradual improvement in clinical symptoms from the fourth day PA through the experiment's conclusion (three weeks-PA). The lack of irritation, bleeding, scale formation, restlessness, and the appearance of smooth skin and new hair growth were signs of the recovery54. The ivermectin-treated animals, on the other hand, gradually improved but did not completely eradicate the condition from the seventh day PA till the investigation's conclusion  demonstrated a slight cellular infiltration, a lack of mites, an increase in the number of hair growth follicles, and the appearance of normal sebaceous glands are signs of progress in the skin's surface layers Fig. C. The skThe-pro-inflammatory-cytokines-,-the-pleiotropic-cytokine-(IL-10)-and-the-monocyte chemoattractant-protein-1-(MCP-1) were all downregulated in the animals treated with mandarin peel oil (20% peel oil in paraffin oil), according to the results of q-PCR. On the other hand, 2–sevenfold increases in ICAM-1, MMP-9, VEGF, KGF, and TIMP-1 were seen Fig. .Figure 7The methodologies of molecular docking intend to predict the best binding orientation of a ligand to a receptor. It proposes several suitable poses of the ligand within the active or docking site of a receptor molecule.The created PPI network comprised of 296 nodes and 1725 edges, which is illustrated in Figs. cytoHubba plugin Cytoscape is considered a useful exploring interface for the most important nodes in the PPI networks, it used to determine the hub genes using ranking methods ,The results shown in  is considered a computational bioinformatic model of biological systems, beginning with the molecular level reaching to the organism level, GO aims to provide knowledge about the functions of gene products, namely, proteins and non-coding RNA molecules. GO is organized in three aspects. GO Molecular Functions (MF) describe activities that occur at the molecular level, Biological Processes (BP) represent the larger processes or ‘biological programs’ accomplished by multiple molecular activities and Cellular Components (CC) which are the cellular structures in which a gene product performs a function, the specific genes expressed in each cell define the identity and functionalities of that cell. Regulation of transcription is highly complex and leads to differential gene expression in specific cells or under specific conditionshttp://www.rcsb.org/pdb/,code 6Y8M). The ligand was re-docked in an active pocket at an acceptable RMSD of 1.311 and an energy score of -5.870 kcal/mol in five interactions of hydrogen bonds along with one ionic bond interaction to verify the results of our research. The involved amino acid residues in the-H-bond-interactions were Thr 147, Met 148, Gln 149, and-Arg 11 as H-acceptor, and another one with-Met-148 as-H-donor, while the ionic interaction was encountered with Arg 11. The-dock-score-of-the-twenty compounds against 6Y8M is summarized in Tables 16 (1-decanol) achieved approximately similar energy score of  − 5.625 kcal/mol when compared with the co-crystalized ligand showing three hydrogen bond interactions as H- acceptor through the hydroxyl group of 1-decanol and the amino acid residues Gln 149,Thr 147 and Arg 11 which resemble the interactions of the-co-crystalized-ligand  complexed with its ligand was obtained from the Protein Data Bank  (tartaric acid). The co-crystallized ligand (l-( +)-tartaric acid) posture was predicted by the docking method with an RMSD-of-1.758-and-an-energy-score-of  − 4.191 kcal/mol. In the style of contacts shown in Table 15 and 16 exhibited better affinity towards the binding site of IL-6 than the ligand, as they showed ΔG of  − 4.372 and  − 4.401 kcal/mol respectively. The hydrogen bond interactions appeared as three hydrogen bond acceptors with amino acid residues Arg 179 and Arg 182 in both, which match the interaction pattern of the co-crystallized ligand, moreover, compound 8 also achieved a good energy score of  − 4.151 kcal/mol when compared with the ligand score of  − 4.191 kcal/mol.  provided the X-ray crystallographic structure of (TNF-) complexed with its ligand. It was demonstrated that the co-crystallized ligand was associated with 16 residues of amino acids and attached inside a small pocket, with seven of those residues coming from-chain-A and the remaining nine-from-chain-B, including-six-tyrosine-residues, from each subunit of the TNF- dimer. This inhibitor works by attaching to the cytokine's active trimer form, stimulating its dissociation into the inactive dimer form, and stabilizing it60. The ligand was re-docked in the active pocket to verify our research. During interactions with receptors, the ligand established hydrogen-bonds-with-Gln-61 as an H-donor-and-with-Tyr-119 as a pi-H interaction. The co-crystallized ligand pose was predicted by the docking method with the least RMSD and an energy score of  − 6.923 kcal/mol. Figure The Protein Data Bank  has-the-X-ray-crystallographic-structure-of-Drosophila melanogaster's delta-class-GST. When glutathione was redocked, it revealed four hydrogen bond interactions, two of which included Arg-67-and-Ser-66 as H-acceptors and the-other-two-involving Glu 65-and Ile 53-as H-donors. The ligand's energy score was  − 5.945 kcal/mol in addition to the two ionic interactions with Arg 67 and Glu 65  of compounds, which play a vital role in their pharmacological activities, especially at the early stages of screening for lead compounds. Consequently, the measurement of these parameters is of great value in the selection of an efficient drug candidate. Lipinski and Veber’s rules are successful tools to perform such screening, as Lipinski’s rule of five states that a compound has drug-like activity if at least three of the following criteria are achieved: a molecular mass less than 500 Da, a maximum of five hydrogen donors, a maximum of 10 hydrogen bond acceptors, and a partition coefficient between octanol and water (LogP (o/w)) smaller than 5. According to Veber’s rule, a compound is orally active if it has 10 or fewer rotatable bonds and a polar surface area (PSA) greater than 140 Å. For predicting drug-like properties, we used Reaxys. The screening of compounds les Fig. .Figure 161. In comparison to conventional drugs, EOs are less likely to cause resistance due to their multiple active components61. EOs may have antibacterial, anti-inflammatory, and antipruritic properties in addition to their scabicide properties14. All these adjuvant properties are especially appealing for the treatment of scabies.Essential oils (EOs) are generally well tolerated, as evidenced by their widespread use in food, hair, and skin preparations62. All these elements work together to make treatment ineffective, especially given that most synthetic medications kill mites rather than altering the immune system or promoting tissue repair. In light of this, plant-derived phytochemicals can operate as safe substitutes for synthetic options for the eradication of infectious diseases due to their broad therapeutic potential and negligible adverse effects63. Citrus fruits have reportedly been found to have immunostimulatory, anti-inflammatory, antimicrobial, and antioxidant properties66. It had considerable antibacterial efficacy against S. aureus and Candida-skin-infections, including-oral-and-vaginal-candidiasis70. Fascinating studies have shown that Citrus oil may change the way that inflammatory responses are expressed, suppressing pro-inflammatory cytokines and enhancing skin's defensive barriers71. No investigation has yet been conducted, to the best of the information we have, on the acaricidal-potential-of mandarin peel oil against Sarcoptes scabiei.As a result of mites burrowing deeply into the skin, scabies pathogenesis is complicated and involves a number of mechanisms, including parasite persistence, which has an impact on both the structure and function of skinSarcoptes scabiei mites 24 h after application. The animals' skin began to exhibit healthy symptoms after the mites died, including the cessation of inflammation-and-hyperkeratosis, the emergence of new-skin-layers, and-the beginning of new hair growth. This occurred at the same time as reports of the effective treatment of rabbit mange72. This full recovery was seen after 3 weeks, whereas the ivermectin group's healing continued until the completion of the experiment (4 weeks) without leading to full recovery. The histopathological findings revealed that the dermis and epidermis of the treated animals improved, inflammation cells decreased, and mite remains were not present in the skin layers. The death of mites, as well as the absence of inflammation, pruritis, skin damage, and scale formation, are the primary causes of the improvement55. In contrast, the skin layers of the ivermectin group saw gradual alterations throughout therapy, and at the end of the investigation, some inflammatory cells as well as traces of deceased mites were still visible. This could be explained by the potent anti-mite effects of ivermectin as well as the common itching and allergic reactions brought on by topical deltamethrin use, which extends inflammation and causes additional delays the emergence of good indicators73.Therefore, the current study examined the GC/MS makeup of mandarin peel oil and assessed the oil's ability to kill Scabies-mites-in-both in vitro-and in vivo testing. There were no symptoms of skin irritability, inflammation, or unease during or after the application of mandarin peel oil. Our findings showed that orange peel oil could have a substantial acaricidal effect on 74. Any imbalance in the activity of keratinocytes, which is crucial for the control of skin immunological homeostasis, might lead to illness. Our results show that when exposed to live digging scabies mites or their waste products , a significant amount of genes in the skin fibroblasts and keratinocytes change their expression, which further activates other cell types75. Hence, in response to scabies, many other skins cell categories, such as lymphoid cells, endothelial cells, or LCs, and dendritic cells, have complex interactions , which results in the development of inflammatory and oxidative stress states. This could increase reactive oxygen species like H2O2, which leads to lipid peroxidation and negatively affects the skin's structure and permeability. In our investigation, faster clinical and parasitological recovery confirmed the potential antioxidant action of mandarin peel oil by restoring the altered oxidant/antioxidant balance in treated animals to normal. Antioxidants are believed to hasten wound healing by reducing oxidative stress on the wound. They are essential in preventing harm from being done to biological elements like DNA, proteins, lipids, and bodily tissue when reactive species are present31. Because the elevated levels of ROS at the site of injury are the main promoters of collagen disintegration, the breakdown of the extracellular matrix (ECM), a decrease in vascular development and re-epithelialization, and a rise in cytokines that are pro-inflammatory, all of which extend inflammation, an extract with ROS scavenging potential could be a key component of the healing protocol31.Epidermal-keratinocytes-as the-first line-of defense against hazardous external invaders must be used to obtain understanding of the-modulative-effects-of mandarin peel oil on the-pathophysiology-of scabies. To recognize various infections and launch immune responses, keratinocytes generate recognition receptors on their surfaces. These receptors allow them to secrete cytokines, chemokines, and anti-microbial peptides that help attract inflammatory cells76. Clinical signs are not noticed for 4–6 weeks after an individual has been diagnosed with scabies mites. This is since the regulatory T cells (type 1) are induced to produce IL-10, a cytokine with anti-inflammatory properties that is required by humans to prevent inflammatory and autoimmune illnesses77. Furthermore, mite products that sensitize keratinocytes are likely to raise the production of VEGF, which is additionally induced by the mites to raise angiogenesis. The mites raise the flow of blood in the region in order to get the nourishment they require from the food being consumed, which worsens inflammation77. A delayed re-epithelialization of the wound is the result of decreased KGF receptor signaling, which also lowers the rate of proliferation of epidermal keratinocytes along the wound edge. Matrix metalloproteinase (MMP-9) is one of a set of hydrolase enzymes that are expressed in many severe conditions, including wounds, osteoarthritis, ischemia, and viral disorders. The inflammation also significantly increases MMP-9 levels78. Nearly all parasite infections use MMP-9 to remodel tissue, which often slows down the production of ECM molecules including collagen II and aggrecan79. TIMP-1  tightly regulates the biological activities of MMPs, and proteolysis results from an imbalance in the MMPs/TIMPs ratio80. Reversing the activation of this network of interrelated genes may therefore be a useful treatment approach to slow the spread of scabies. When CSE was applied topically, the expression of IL-1, 6, 10, VEGF, MMP-9, and MCP-1 significantly decreased, whereas the expression of i-CAM-1, KGF, and TIMP-1 significantly increased.The anticipated mechanism of action of mandarin peel oil on scabies-infected rabbits was shown schematically in Fig. The outcome was an improvement in host immunity against invading mites, a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory ones, which could reverse the unfavorable symptoms and lead to improved re-epithelialization, rapid recovery, and a decrease in inflammation.With instances of treatment failure and the emergence of resistance, controlling scabies effectively using the available acaricidal medicines has proven to be extremely difficult. With a biocidal performance comparable to that of traditional synthetic treatments, this study demonstrated the mandarin peel oil's acaricidal efficacy against mange mites in rabbits. The work examined the composition of the oil and revealed the presence of different hydrocarbons and their oxygenated forms, with proved biocidal activities. Additionally, the oil has been tested against naturally infected rabbits with mange using different techniques and proved higher efficacy and safety compared to market agents. The mandarin peel oil presents an ideal alternative to commercial medications used for the control of arachnids that can harm humans and animals while being economical, safe, and environmentally friendly. These candidates can be successfully employed to create novel biocides for applications in agricultural improvement and livestock protection.Supplementary Information 1.Supplementary Information 2.


The protein composition and digestive characteristics of four commercially available infant formulae (IF) manufactured using bovine (B-IF), caprine (C-IF), soy (S-IF), and rice (R-IF) as a protein source were examined in this study. Plant-based formulae had significantly higher crude protein and non-protein nitrogen (NPN) concentrations. Static in vitro gastrointestinal digestion of these formulae, and subsequent analysis of their digestates, revealed significantly higher proteolysis of B-IF at the end of gastrointestinal digestion compared to the other formulae, as indicated by the significantly higher concentration of free amine groups. Furthermore, differences in structure formation during the gastric phase of digestion were observed, with formation of a more continuous, firmer coagulum by C-IF, while R-IF demonstrated no curd formation likely due to the extensive hydrolysis of these proteins during manufacture. Differences in digestive characteristics between formulae manufactured from these different protein sources may influence the bio-accessibility and bioavailability of nutrients, warranting additional study. Breastmilk is considered the most suitable form of nutrition for infants, as recommended by the World Health Organisation. However, in circumstances where breastfeeding is not possible or is discontinued, infant formulae (IF) is a suitable alternative to breastmilk to fulfil the infant’s nutritional requirements until such time that complementary foods may be introduced. Protein intake is extremely important for infants, influencing their growth, body composition, appetite, and hormonal regulation . IngrediProtein digestibility is affected by structure formation during digestion, which impacts nutrient bio-accessibility within the gastrointestinal tract (GIT) ,11,12. SA variety of different models and experimental conditions have been used to replicate infant digestion across these studies, with slight but important variations in physiological parameters, such as digestion time, pH, and enzyme activities. To overcome this, a standardised in vitro static digestion method based on an extensive review of current knowledge on infant digestion physiology  and aligin vitro.In this context, the objectives of the current study were to analyse the protein composition and profile of commercially available infant formulae manufactured from animal- and plant-sourced ingredients and to study their digestive characteristics Four commercially available IF powders suitable for infants aged 0–6 months were selected: bovine milk-based IF (B-IF), caprine milk-based IF (C-IF), soy-based IF (S-IF), and rice-based IF (R-IF). All the samples were purchased in a local supermarket in Ireland except the rice-based formula, which was purchased from a supermarket in France. Reconstituted IF samples were prepared in ultra-pure water according to the manufacturer’s recommendations.The total nitrogen content of the reconstituted IF samples was determined using the Kjeldahl method  with 3 mg at 4 °C) for 1 h. TCA (24%) was then added to the supernatant at a ratio of 1:1 to achieve a final TCA concentration of 12%, after which the samples were filtered using 0.45 µm syringe filters  and transferred to glass vials. Samples were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC)  equipped with an Aeris Widepore XB-C-18 column . Gradient elution was carried out with a mixture of solvents containing 0.1% TFA in ultrapure water (solvent A) and 0.1% TFA in acetonitrile (ACN) (solvent B). Separations were performed using the program reported in Reversed-phase high-performance liquid chromatography was performed, as reported by Vogelsang-O’Dwyer et al. , to obta2 was added separately, at a concentration of 3 mM, to the intestinal fluid. The intestinal phase was halted by the addition of 50 µL of 0.1 M Pefabloc® SC , providing time point I60. Samples at the gastric timepoints G0, G30, and G60 were obtained by adjusting the pH of the gastric chyme to pH 8 using 1 M NaOH immediately after its initiation, 30 min, and 60 min, respectively, and then adjusted to 5.3 for further analysis. Digestion was carried out in triplicate for each IF and control with distilled water. Samples collected at each time point were divided into Eppendorf tubes in 1 mL aliquots, blast frozen at −20 °C for 20 min , and stored at −80 °C until further analysis.Static in vitro digestion of each IF was carried out using the method of Ménard et al. , with miThe protein profile of the reconstituted IF and IF digestates were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using pre-cast Mini-Protean Tetra cell TGX 4–20% acrylamide gels  under reducing conditions with β-mercaptoethanol as the reducing agent. Samples were centrifuged at 1000 rpm for 30 min. Aliquots of sample providing a protein load of 10 µg were loaded into the wells of the gels, and electrophoretic separation was performed at a constant voltage of 160 V. The gels were stained using Coomassie blue R250 and de-stained using a solution of water, methanol, and acetic acid at a ratio of 50:40:10, respectively. The stained SDS-PAGE gels were scanned using a desktop flatbed scanner .g for 30 min at 22 °C. The supernatant was filtered using 0.45 µm syringe filters . The OPA reagent used was as described by Nielsen et al. [The degree of protein hydrolysis in reconstituted formulae and digestates was assessed using an O-phthaldialdehyde (OPA) spectrophotometric assay. Reconstituted IF and digested samples were pre-treated with TCA  and centrifuged at 10,000× n et al. . L-leuciThe free amino acid (FAA) composition of the IF digestates at the end of gastrointestinal digestion (I60 samples) was analysed using cation exchange liquid chromatography coupled with post column ninhydrin detection . DeproteThe microstructural properties of the IFs before, during, and after digestion were assessed using confocal laser scanning microscopy (CLSM). Aliquots (400 μL) of the reconstituted formula or digestates were stained with 10 μL of 0.1% Fast Green FCF (aq.) and 20 μL of 0.02% Nile Red , after which samples were transferred to microscopy slides and covered with glass coverslips. A Leica TCS SP5 confocal laser scanning microscope  was used to observe the stained samples using a 63×/1.4 oil immersion objective. Fast green FCF was excited at 633 nm using a He/Ne laser, and the corresponding emission filter was set at 685–750 nm. Nile red was excited at 488 nm using a diode-pumped solid-state laser, and the corresponding emission filter was set at 600–615 nm. The analysis was performed at the Teagasc National Food Imaging Centre .w/v) was added to the samples and stirred vigorously for 30 s. Small amplitude oscillatory rheological measurements were carried out using a Discovery Hybrid 2 Rheometer  fitted with a concentric cylinder geometry at 30 °C. A time sweep analysis was performed using the following conditions: 5 s temperature equilibration at 30 °C, 15 s pre-shear at 50 s−1, and 10 s equilibration, followed by oscillation at 0.1% strain at a frequency of 1 Hz. A second treated IF sample remained in the adjacent water bath at 30 °C, the pH of which was monitored continuously until pH 4.6 was reached, at which point the time sweep was stopped. The gel point was defined as the point at which the value for the storage modulus (Gʹ) was >1 Pa.The coagulation properties of IFs under acidic conditions were determined using a method previously described by O’Callaghan et al. , with mop < 0.05. Multivariate statistical analysis was carried out on the results of the free amino acid analysis using MetaboAnalyst software , from which Statistical analysis was performed using IBM SPSS Statistics V28 . Statistical differences between the means of experimental data from N analysis, FAA composition, and the acid coagulation properties of IF were assessed by analysis of variance (ANOVA), followed by Tukey’s posthoc analysis. Analysis of the OPA assay results was carried out using repeated measures ANOVA with posthoc Bonferroni adjustment. Results were considered statistically significant when The nitrogen and crude protein content of the plant-based formulae was found to be significantly higher than the dairy-based formulae . Plant pThe NPN fraction is diverse in terms of nitrogenous compounds, which remain soluble in 12% TCA . While tThe concentration of NPN was significantly different across the different formulae, and R-IF > S-IF > C-IF > B-IF when expressed as a % of total N , which iThe concentration of NPN in S-IF was higher than that in B-IF and C-IF at 0.07%, making up 24.6% of the total nitrogen content in these formulae. This is in agreement with findings by Donovan and Lonnerdal , who repThe concentration of NPN was considerably higher for the rice protein formulae at 0.21% for R-IF, corresponding to over 86% of the total nitrogen content in this formula . This maThe generally poor solubility of plant proteins ,42, and S1-casein in goat’s milk. The band for β-lactoglobulin at 18 kDa was correspondingly larger in B-IF than in C-IF, also potentially reflecting the difference in their casein to whey ratio. Additionally, the more distinct band for β-casein in C-IF indicates dominance in this formula, as would be expected for caprine IF, in contrast to bovine milk, where αs1-casein is the dominant casein [t casein .The proteolytic action during the gastric phase of digestion was clearly evident in the electrophoretograms of B-IF and C-IF A,B. AfteIn contrast to the caseins, the bands representing the whey proteins remained intact throughout gastric digestion. The resistance of whey protein to in vitro gastric digestion has been widely reported ,19,25,48The electrophoretogram for S-IF C displayThere was a significant change in free amine groups in each sample as digestion progressed from reconstituted samples through to G60 and I60 stages, as expected; however, the levels of free amine groups in R-IF remained relatively stable between G0 and G60 timepoints as is shown in The results shown in Furthermore, B-IF was found to contain a higher concentration of free amine groups than C-IF after 60 min of intestinal digestion, suggesting more extensive proteolysis of B-IF at this point. Few studies have compared the digestion of infant formulae manufactured from bovine and caprine milk protein, with most focusing on the gastric phase of digestion rather than the intestinal phase ,18. Zhoup < 0.05) [Using a dynamic in vitro model replicating infant digestion, Maathuis et al.  reported < 0.05) . However < 0.05)  in theirr = −0.98) between the proportion of β-sheets in the secondary structure of a variety of proteins and their values for digestibility. This is explained by the high degree of hydrophobic interactions within the β-sheet structure, which are associated with protein-protein interaction and the formation of protein aggregates, particularly after heat treatment, which can limit the digestibility of the soy protein [S-IF exhibited a lower degree of hydrolysis at I60 than B-IF . Similar protein ,57.In spite of the high degree of hydrolysis observed in the reconstituted rice protein formula before digestion, B-IF exhibited a higher degree of proteolysis than the rice protein-based formula after 60 min of intestinal digestion . While tp < 0.05) in the concentration of cysteic acid between B-IF and C-IF, S-IF, and R-IF.The free amino acid (FAA) composition of the intestinal digestates (I60) is given in p < 0.05), and there was a significant difference between B-IF and C-IF, B-IF and R-IF, C-IF and R-IF, and S-IF and R-IF. Serine concentrations of S-IF and R-IF were significantly higher than that of C-IF. Alanine concentrations were significantly higher in R-IF than that in B-IF, C-IF, and S-IF.There was no significant difference in the concentration of cysteic acid between R-IF and S-IF. Threonine concentration was significantly higher in R-IF than in other formulas (p < 0.05), while the leucine concentration of C-IF was significantly lower than that of B-IF and R-IF. Phenylalanine levels were significantly lower in C-IF than in other formulae (p < 0.05), and B-IF had significantly higher levels of lysine than C-IF and S-IF. S-IF had significantly lower tryptophan than that of C-IF and R-IF.Valine concentration of the intestinal digestates was significantly lower in C-IF than that of B-IF and R-IF. B-IF contained significantly more free methionine than the C-IF, S-IF, and R-IF . Isoleucine concentration of the R-IF digestates was significantly higher than that of B-IF, S-IF, and C-IF  demonstrated in CLSM images for the infant formulas at each time point of digestion are presented in During digestion, microstructural changes were also evident among the formula samples. The images for B-IF and C-IF show the progressive formation of aggregates and coagulation of proteins from the early stages of gastric digestion, especially for the latter, for which coagulation can be observed as early as in sample G0. This was likely due to the decrease in pH to 5.3 during the initiation of the gastric phase of digestion, which approached the isoelectric point of the caseins (pH 4.6), more abundant in C-IF than in B-IF samples. The coagulation of dairy proteins at the early stages of digestion is widely reported  and depep < 0.05). Within the dairy-based formulae, C-IF reached a significantly higher complex modulus (G*) value at pH 4.6 than B-IF . This is also likely to be due to the higher casein–whey ratio of C-IF, resulting in the formation of increased crosslinks between micelles leading to stronger gel networks. This was also reported by Wang et al. [s1-casein and larger casein micelles in goat milk compared to cow’s milk, leading to the formation of a softer, more fragile curd when products with the same whey-to-casein ratio produced from these milks are compared [The protein source from which infant formula is manufactured was shown to have significant effects on its acid gelation properties. The results presented in g et al. , where ug et al. . This wacompared ,47,63. Tcompared .Similar to the lack of structure formation observed for plant protein-based formulae in the CLSM micrographs of their digestates , these fThe results of the present study have demonstrated differences in the protein composition of dairy- and plant-based infant formulae, while there are limitations to this study given the wide variety of commercial formula and formats of ingredients available on the market today between fresh milk versus powdered ingredients, hydrolysed and unhydrolyzed ingredients sources. Nevertheless, in vitro digestion of these formulae has demonstrated that the source of protein used in the manufacture of infant formula significantly impacts its digestive properties in terms of both structure formation within the gastric phase of digestion and overall digestibility. As such, this may imply altered digestive behaviour when consumed by an infant, which could impact the bioaccessibility and bioavailability of nutrients from these formulae, and therefore, these results may have implications for the optimisation of infant formulae composition and warrant further study.


The structural similarity index measure, mean squared error, and peak signal-to-noise ratio of predicted images improved significantly (p < 0.0001). The resolution of predicted images presented close values to ground truth images (0.18 mm and 0.15 mm versus 0.15 mm). The gingival thickness measurement showed a high level of agreement between the predicted sparse array images and the ground truth images, as indicated with a bias of -0.01 mm and 0.02 mm for the 64- and 16-channel predicted images, respectively, and a Pearson’s r = 0.99 (p < 0.0001) for both. The gingival thickness bias measured by deep learning assisted sparse array imaging and clinical probing needle was found to be <0.05 mm. Additionally, the deep learning model showed capability of generalization. To conclude, the deep learning assisted sparse array can reconstruct high-resolution ultrasound image using only 16 channels of 128 channels. The deep learning model performed generalization capability for the 64-channel array, while the 16-channel array generalization would require further optimization.This study aims to restore grating lobe artifacts and improve the image resolution of sparse array ultrasonography via a deep learning predictive model. A deep learning assisted sparse array was developed using only 64 or 16 channels out of the 128 channels in which the pitch is two or eight times the original array. The deep learning assisted sparse array imaging system was demonstrated on  Two-dimensional 2D) and three-dimensional 3D) ultrasonography has been widely applied in imaging tissues and organs for diagnosing diseases due to its capability of serving as a real-time, non-invasive, portable, and radiation-free tool . UltrasoD and thrD ultrasoSparse array ultrasound imaging has been reported to reduce the number and density of the transducer elements, including random array , non-uniDeep learning methods have been applied in ultrasound imaging for automatic organ segmentation , object in vivo/ex vivo experiments were performed but with a pitch reduction of only two-fold  architecture was presented to restore the undersampled sparse array imaging with significant grating lobe artifacts . The RUNIn this study, the performance of the proposed CNN-assisted sparse array imaging was demonstrated on porcine teeth because of the value ultrasonography can have in oral health –27. The Assisted with the RUNet model, a high-resolution sparse array imaging system has been demonstrated. Only 64 channels or 16 channels out of the total 128 channels were activated, and the pitch of the sparse array is eight or two times that of the original dense array. To the best of our knowledge, this investigation is the first to report on the use of an eight-fold pitch for sparse array ultrasound imaging, and it is also the first to apply the sparse array technique in periodontal applications.The CNN model was trained using 64- or 16-channel sparse array images as the input and 128-channel dense array images as the ground truth . The performance of the CNN model was evaluated based on the improvement in image quality and the accuracy of landmarks localization accuracy of the predicted images. Please see the subsequent sections for more detailed information.Ultrasound transducer L35-16vX  was used to collect ultrasound images of periodontal structures. The transducer has a linear array with 128 individual channels . By regularly turning off every other channel of the original 128-channel transducer, the 64-channel sparse array transducer has a pitch of 140 μm. Similarly, by regularly turning off seven out of eight channels of the original 128-channel transducer, the 16-channel sparse array transducer has a pitch of 560 μm. A 30-μm nichrome wire was immobilized in water under 10 mm from the surface of the transducer for characterizing the axial and lateral resolution of the B-mode imaging by the point spread function full width at half maximum (FWHM)  29]. Th. Th29]. A data acquisition (DAQ) system  was connected to the transducer to provide a 15-V power supply, generate pulses with a repetition rate of 5 kHz, sample the radiofrequency data of each channel, and reconstruct ultrasound images  Figs. ThB-mode ultrasound imaging with a size of 512 pixels × 512 pixels was performed with coherent compounding beamforming by applying a series of time-delayed electrical excitation in each transducer channels . HanningFresh porcine jaws were purchased from a local abattoir and prepared as previously reported . The porThe RUNet is composed of two sections of dimensionally symmetric convolutional layers: encoder and decoder . The encA collection of 1500 cross-sectional images with a size of 512 × 512 pixels from 10 teeth of three porcine jaws was obtained using three different transducer channel configurations . At the same position of the same tooth, one image was captured using each of the three transducer channel configurations. As a result, the image dataset consists of 500 image pairs each pair containing one 128-channel image, one 64-channel image, and one 16-channel. Of the 500 image pairs, 400 pairs were divided into training (80%) and validation (20%) datasets. The images captured with the 64- and 16-channel transducers were labeled as sparse array inputs, while the images captured with the 128-channel transducer were labeled as ground truth. The remaining 100 image pairs served as the test dataset. The test dataset also contained an image of a tooth from a previously unseen fourth porcine jaw, images of the radial artery and thenar muscles, and images of noises. The inclusion of a tooth image from a new porcine jaw could evaluate the generalization capability of the predictive model. The images of radial artery, thenar muscles, and noises were collected as negative control to investigate the over-fitting possibility of the deep learning model .The images were manually analyzed and measured. Before collecting the 64- and 16-channel sparse array images, the examiner had to ensure that all 128-channel ground truth images met specific quality criteria, which included: (1) identifying the GM and gingival surface, (2) identifying the tooth surface, and (3) ensuring there were no interfering artifacts coincident with the relevant anatomy. If these conditions were met, further image collection was performed.The original images were compared to the reconstructed images. This comparison used established metrics known as the structural similarity index measure (SSIM), mean squared error (MSE), and peak signal-to-noise ratio (PSNR). These metrics were measured in MATLAB to quantitatively compare the image quality of the input sparse array images with the images predicted by the CNN model. SSIM was used to measure the structural similarity between two images . SSIM raGT (σGT) and μCNN (σCNN) represent the pixel sample mean (variance) of the ground truth images and CNN input/predicted images, respectively. σcov is the covariance of ground truth images and CNN input/predicted images. k1 and k2 are two variables to stabilize the division with a weak denominator.Here, μMSE calculated the corresponding pixel value differences of two images . MSE ranGT and ICNN are the ground truth and CNN input/predicted images of sizes of M×N, respectively.Here, IPSNR measures image quality by comparing the maximum possible power of the original image to the power of the noise introduced during compression or processing . Higher max indicates the maximum possible values in the given images.Here, IThe gingiva region was manually segmented with ImageJ by drawing the boundary along the gingiva surface, the GM, and then the tooth surface on a 28-inch monitor with resolution of 3840 pixels × 2160 pixels . The IoUGingival thickness was measured using ImageJ software from the reconstructed ultrasound images . For conIn the clinical transgingival probing method, a 28-gauge needle with calipers was inserted into the gingiva perpendicularly through the gingival surface until the needle touched the tooth surface . By markA power analysis was conducted using G*Power to estimate the adequacy of the sample size (n) for determining differences in gingival thickness measurements between images grouped as 128-ground truth vs. 64-predicted and 128-ground truth vs. 16-predicted, respectively, using a two-tailed significance test with a 95% significance (α = 0.05), and minimum differences of 0.3 mm, 0.5 mm, or 1.0 mm . Each ofBland-Altman analysis was performed to characterize the bias and limits of agreement between measurements of grouped predicted images and ground truth images, and between two image examiners. Pearson correlation was performed to quantify the agreement between grouped images. A paired t-test was applied to justify if the measurements of grouped images were significantly different. A p-value < 0.0001 was considered significant. Bland-Altman analysis, Pearson correlation, and paired t-test were performed using GraphPad Prism version 9.5.0 .th pre-molar) reconstructed from the 128-channel transducer was shown in A representative image of porcine tooth  had sufficiently high power (≥ 80% by convention) to detect a minimum difference of ≥ 0.5 mm . FinallyNext, as a test of practical utility and generalization, the deep learning algorithm was used to image a porcine tooth (1st molar) completely separate from the training dataset . The visFinally, a group of ultrasound images not from tooth were input as the test dataset to serve as negative control, including images on radial artery , noise , and theAccording to the Huygens principle, the pitch of linear array ultrasound transducer is required to be close to one wavelength to form a single wave front from wave fronts from each element . When thIn the 64-channel sparse array imaging, the grating lobe artifacts reduce the resolution, which leads to inaccurate anatomical feature localization. The ABC cannot be distinguished from the background, and the tooth surface below the CEJ is merged with the artifacts. There are no detectable anatomical features in the 16-channel input image due to the huge pitch that was of ten times of the transducer wavelength. The RUNet predictive model can eliminate artifacts and differentiate the anatomical features from the background. The quality of the 64- and 16-channel output images predicted by the algorithm is comparable to the ground truth images reconstructed with the 128-channel transducer as indicated by the close resolution of tooth surface (0.18 mm for the 64-channel and 0.15 mm for the 16-channel vs. 0.15 mm for the 128-channel). The SSIM, MSE, and PSNR significantly improved (p < 0.0001) when comparing the predicted images to the input sparse array images, showing the considerable image quality improvement by the CNN model.Additionally, the accuracy of morphology in the predicted images was characterized by measuring a clinical periodontal index: the gingival thickness. Based on an ex vivo study conducted on 20 porcine cadavers, Kloukos et al. reported that clinical transgingival probing can be considered as reliable for assessing gingival thickness values . Compariex vivo porcine teeth as a training dataset to train the CNN model. One can then possibly apply the well-trained model to human subjects. In this way, redundant efforts in collecting hundreds of datasets in human subjects could be avoided.In the generalization test using a separate porcine tooth as the input, the reconstructed shape of anatomical structures in the 64-channel predicted image are consistent with that of the 128-channel ground truth image, as indicated by the high IoU of the gingiva region (0.97). The generalization capability of 64-channel imaging provides potential for using the The 16-channel predicted image could reproduce the anatomy of the gingiva and tooth in a generalization test but with reflection signals below the GM. These reflections are not seen in the 128-channel ground truth images. Also, the gingival surface morphology is not consistent with the ground truth image. The 16-channel input sparse array images are more undersampled than the 64-channel. Thus, it is more challenging for the CNN model to generalize the 16-channel prediction effectively. Clinically, a 16-channel sparse array is probably unlikely to be needed. Transfer learning could be applied to the unseen new subjects and/or further enlarge the training dataset to improve the generalization of 16-channel sparse array.The RUNet CNN model applied in this study was trained by supervised learning, which required paired low-resolution and high-resolution images for training. However, obtaining paired data could be challenging and impractical in clinical settings. In 2017, Ledig et al. reported a super-resolution GAN model that could generate photo-realistic super-resolution results . The GANThis study reports a deep learning-assisted sparse array ultrasound imaging system to reduce the cost of fabrication, complexity of channel control, and electrical power consumption. The proposed sparse array system only requires 1/8 of the traditional ultrasound transducer channels to generate high-resolution images that have comparable quality of the original dense array.The SSIM enhances from 0.66 to 0.73 when using 64 out of 128 channels and from 0.56 to 0.68 with using only 16 out of 128 channels. The resolution of the sparse array image predicted by the CNN model is close to that of the original dense array (0.18 mm for the 64-channel and 0.15 mm for the 16-channel versus 0.15 mm for the 128-channel). The deep learning model shows promising generalization capability for use in more subjects/sites. Negative controls proved that the model did not over fit during the training process. Future work will include improvements in device instrumentation . Additional optimization of increasing the generalization capability will include enlarging the datasets to more diverse imaging targets and introducing transfer learning in the deep learning algorithms. Besides imaging the tooth anatomy, the sparse array imaging application could be further expanded in imaging different tissues and organs  to evaluate the cardiovascular diseases. Clinically, the sparse array imaging could be transferred from porcine teeth to human subjects  for clinical translation.S1 Fig(a) Wire imaging reconstructed from 128-, 64- and 16-channel ultrasound probe. (b) Characterization of axial (left) and lateral (right) resolution of the 128-, 64- and 16-channel probe.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe system includes ultrasound transducer and DAQ system. The ultrasound transducer is connected to DAQ system for power supply, data processing and real-time imaging display.(TIF)Click here for additional data file.S4 FigRed line: 2-mm from the GM. Yellow line: gingival thickness. GM: gingival margin.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigThe scale bar applies to all images.(TIF)Click here for additional data file.S7 FigThe scale bar applies to all images.(TIF)Click here for additional data file.S8 Fig(a) 128-ground truth, (b) 64-predicted, and (c) 16-predicted images.(TIF)Click here for additional data file.S9 FigMean differences were 0.3 mm (red), 0.5 mm (blue), and 1.0 mm (green). Effect size was calculated using gingival thickness measurement standard deviations of (a) 128-ground truth vs. 64-predicted and (b) 128-ground truth vs. 16-predicted, respectively.(TIF)Click here for additional data file.S10 FigSSIM: structural similarity index measure.(TIF)Click here for additional data file.S11 Fig(a) In the new tooth from a new porcine jaw, the IoU of gingiva region was 0.97 for 128-ground truth vs. 64-predicted and 0.73 for 128-ground truth vs. 16-predicted. (b) In one of the ten teeth in the training dataset, the IoU of gingiva region was 0.96 for 128-ground truth vs. 64-predicted and 0.96 for 128-ground truth vs. 16-predicted. Yellow, blue, and red regions indicate the gingiva region of 128-ground truth, 64-predicted, and 16-predicted image, respectively. Green and orange regions indicate the intersection of the 128-ground truth with the 64-predicted and 16-predicted, respectively. IoU: intersection over union.(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images. Three-dimensional 3D) volumetric imaging is important for understanding the structural and functional characteristics of tissues and organs. The nervous system represents the most complex sample because individual neurons extend in long directions to many brain regions, and several neuronal groups compose neural circuits. Morphological studies on whole-brain imaging can explain the intrinsic and physiological brain functions by obtaining the population, altered morphology, and number of various cell types ,2. HowevD volumetConfocal microscopy produces 3D volumetric images with high signal-to-noise ratios through the reconstruction of a sectioned image of up to a couple hundred microns in size into cleared samples ,8. Thus,In order to obtain a suitable 3D image of a cleared whole mouse brain, we compared the commercially available Dragonfly spinning-disk-type confocal microscopy  with Lightsheet Z.1 , which was changed to a 2.5× objective lens and chamber, a modified sample holder of Lightsheet Z.1. This paper investigates the basic concept for suitable microscopy applications for whole mouse brain imaging and the most suitable way to acquire quick, wide, and deep images with high resolution.The genetically modified Tg(Thy1-EGFP)MJrs/J mouse line was kindly provided by the laboratory of Dr. Rah in KBRI, which was originally purchased from Jackson Laboratories  and bred in-house. The Tg (Thy1-EGFP) MJrs/J mice were genotyped with a primer set from the JAX protocol . The genA 4% PFA-fixed Thy-1-GFP mouse brain was cleared using the Rapid™ tissue-clearing method . The procedure followed the manufacturer’s company protocol. Briefly, the fixed mouse brain was cleared into the Binaree Tissue-Clearing RapidTM Chamber, which is an electrophoresis chamber, for 4 h. Next, the cleared whole brain was transferred into a Binaree mounting solution and agitated at 50 rpm/37 °C for 24–36 h. Subsequently, the reflex index (RI) was matched to the 3D whole-brain image and imaged using Dragonfly spinning-disk-type confocal microscopy and Lightsheet Z.1 microscopy. The normalized expansion ratio of the initial state to the final distortion (at 36 h) was calculated for quantification.We used the Lightsheet Z.1  microscope from Zeiss for our imaging experiments. To enable whole mouse brain imaging, we applied an imaging chamber  to a microscope with a 2.5× objective lens . The imaging chamber was approximately 42% larger than the chamber in the original Lightsheet Z.1 model. For the sample preparation, we glued a whole mouse brain to the bottom surface of the modified mounting holder. For the imaging process, we used a 2.5× objective lens  and a 5× illumination lens  with a 488 nm excitation laser source . The data acquisition was performed using 10% laser power as the acquisition condition, and the camera  settings included an exposure time of 30 ms. Pixel size is 2.57 µm × 2.57 µm. All acquisitions were controlled by the ZEN software . For the fast-confocal microscope, we used the Dragonfly 502 w model . The objective lens used was a 4× lens  with a 488 nm excitation laser source. . The data acquisition was performed using 10% laser power as the acquisition condition, and the camera  settings acquired an exposure time of 150 ms. Pixel size is 3.01 µm × 3.01 µm. All acquisitions were controlled by the Fusion software . The samples were imaged using an inverted microscope setup on a glass-bottom dish.To address the limitations of the existing sample holder, which caused dead volume at the bottom of the sample due to the chamber structure and stage movement range of the Lightsheet Z.1 system, we designed a detachable cap using a J826™Prime 3D printer ). This cap matched the size of the dead volume, allowing for compatibility with the existing sample holder.® Silver 4314 CPU 3.4 GHz, 128 GB RAM, Windows 10 x64 bit, and NVIDIA RTX A6000 GPU.The images from both microscopies were rendered to 3D images using the Imaris software  and further analyzed. All files were converted into an ims format, and manual stitching was performed using the Imaris stitcher . Additionally, ImageJ was used for a sample expansion ratio analysis, and the Origin software  was used for histogram and Gaussian fitting. The workstation used for the analysis and post-processing had the following specifications: Intel XeonTo verify the spatial resolution of the microscopes at low magnification, 4μm diameter fluorescent beads  were utilized, and their FWHM of the point spread function was measured through Gaussian fitting. In the FCM case, measurements were taken by simply diluting the beads, applying them to a glass slide, and then measuring them. For LSFM, the beads were embedded in 2% agarose, fixed to sample mounts, and immersed in a matching solution for measurement.To acquire optical 3D volume imaging of a specimen larger than a few centimeters, tissue-clearing methods are necessary. We used the Rapid™ tissue-clearing method (Binaree) to achieve transparency, which enabled imaging with light-sheet and fast-confocal microscopy. This method employed an electrophoresis step to accelerate the tissue-clearing process and allowed for the rapid clearing of whole mouse brains within a few hours. The cleared brains were placed in a Binaree-manufactured mounting solution, which is a high-RI matching solution A. DuringIn order to image a whole mouse brain, a system capable of imaging a wide FOV is required. Thus, researchers need to choose an image of appropriate resolution, identify the image from the whole brain, and consider the speed of image acquisition and data handling for multi-scale images. The spinning-disk-type confocal microscope is known for its relatively faster speed compared to traditional confocal laser-point scanning microscopes. Here, we used Dragonfly spinning-disk-type confocal microscopy as the fast-confocal microscope (FCM) and compared it with the LSFM. LSFM has the advantage of obtaining high-speed, large-scan images owing to its ability to acquire a single slice of sample at a time using a light sheet. Lightsheet Z.1 has a Z-axis limitation between the objective lens and the single light-sheet plane of the illumination lens A. AdditiSubsequently, we compared the 3D volume images and resolution of both microscopes and identified that FCM had a limited imaging depth compared to LSFM, reaching around 3 mm deep before encountering difficulties in acquiring data, whereas LSFM enabled imaging of the entire brain A,B,E,F. Although high-resolution 3D images are typically obtained using high-magnification FCM lenses, the wide-field imaging of large tissue areas is still time-consuming. By using low-magnification lenses to achieve a wider FOV, the Z-resolution is inevitably compromised. Most researchers are aware that confocal microscopy acquires high-resolution images with high signal-to-noise ratios using single-photon line scans. Thus, we performed a comparison between the images acquired via LSFM and FCM, and the fluorescent intensity with depth was acquired via LSFM B. We perIn this study, for the first time, we compared FCM and LSFM for whole mouse brain imaging using a rapid tissue-clearing method by the Binaree Tissue-Clearing RapidTM system. As a comparison, we used the Dragonfly 502 w model as the spinning-disk-type FCM, which is known to be suitable for fast-scan imaging and large-volume imaging. Additionally, we applied the specifically modified Lightsheet Z.1 as the LSFM for whole-brain image acquisition by using an expanded chamber and low-magnification lenses for wide FOV acquisition. We also addressed the limitations of the specimen holder by designing a detachable socket, which allowed for imaging the whole mouse brain without a dead volume area.FCM showed a significant difference in acquisition speed compared to LSFM, providing a 3/4 field of view (FOV) compared to LSFM but demonstrating convenience in the pre- and post-acquisition processes. On the other hand, LSFM required careful consideration of specimen exposure during the specimen mounting and potential damage from glue-chip formation and detachment. When comparing the image data from these two types of microscopes, it would have been ideal to use the same magnification as the 2.5× lens used in LSFM. However, we used the 4× lens for the confocal microscope because higher-resolution images could be obtained. Therefore, the total process time in In the case of FCM, a cleared brain with a thickness of up to approximately 500 μm can be easily viewed using a standard 10× lens, and high-resolution imaging is possible depending on the working distance of the lens. However, for samples with a thickness greater than 500 μm to up to a few millimeters, lower resolution with lower-magnification lenses might have to be applied.The specimen mounting time of LSFM is essential and considered a critical factor in the imaging process, and with a specimen thickness ranging from hundreds of micrometers to approximately 2 mm, securing the sample with glue can be challenging. Thin samples, such as skin tissues, are challenging to image, particularly because simple glue fixation can lead to fluidity within the chamber solution . AgaroseNumerous tissue-clearing techniques are available; however, we used the Binaree system, which removes whole mouse brain lipids by electrophoresis within a few hours. When we use this system, the whole brain was expanded approximately 2.5–3 times larger compared to its original size and shrinks to around 1.6–1.8 times after RI matching. Although the volumetric size of the whole mouse brain was magnified by the tissue-clearing method, LSFM took a high-resolution whole-brain image with an 8.3 mm Z-depth, whereas FCM took a half-brain image with a high-resolution that reached around 3 mm. Hence, researchers must choose a microscope system based on the size, thickness, and transparency of their sample. However, there is considerable confusion about whether there is an error in the tissue-clearing process or if there is a limitation with the microscope, and the problem can worsen if researchers assume that confocal microscopy automatically provides superior resolution or that LSFM is suitable for large-sample images.In this study, we found that Lightsheet Z.1 is suitable for whole mouse brain imaging by using a modified objective lens, a solution chamber, and a sample holder socket compared to Dragonfly spinning-disk-type confocal microscopy. The modified Lightsheet Z.1 represents the one of advantage of Lightsheet 7 for large-volume imaging at a relatively low price. Taken together, these results provide useful information to researchers about the most suitable type of microscopy for large-volume images by considering factors such as entire specimen size, distribution and size of target proteins, acquisition time, and large data analysis.


Although various total knee arthroplasty (TKA) implant designs are widely used, the ideal TKA design is yet to be agreed upon. Although the benefits of cruciate-retaining (CR) TKA and medial stabilized (MS) TKA have been reported in literature, the early clinical outcomes of an MS TKA with CR inserts have not been reported. This study aims to report on the patient-reported clinical and radiological outcomes of MS-TKA combined with a CR insert.A prospective single-surgeon series evaluated the clinical- and patient-reported outcomes of 115 patients implanted with GMK Sphere CR. Patient outcomes were assessed with the Oxford Knee Score, Knee Injury and Osteoarthritis Outcome Scores (KOOS), Forgotten Joint Score, and Visual Analogue Scale for Satisfaction. Radiological assessment for alignment along with active flexion and extension were also assessed.P = .021 and P = .001).Improvement in all scores was observed between the preoperative and 1-year follow-up timepoints, with statistical significance seen for Oxford Knee Score as well as KOOS Symptoms, Pain, Sport, quality of life, and activities of daily living subscales. The mean active flexion between the preoperative and both postoperative timepoints at 6 months and 1 year was also statistically significant (MS-TKA with a CR insert can facilitate symptom relief and improve overall function of the knee after surgery. Both the patient and clinical outcomes were comparable to 1-year outcomes utilizing other MS-TKA designs and were superior to those at 1-year follow-up following implantation of CR-TKA. Most notability, the KOOS symptoms and sports score were higher for the MS-TKA with a CR insert than for an MS-TKA design. Total knee arthroplasty (TKA) has revolutionized the treatment of patients with degenerative arthritis of the knee. Since its invention, the design of TKA has undergone significant revision and modification, with new implant designs and refinement of surgical techniques leading to improvements in clinical outcomes and survivorship rates ,2. HowevThe cruciate-retaining TKA (CR-TKA) centers around posterior cruciate ligament (PCL) retention with the assumption that preserving the native PCL is advantageous in aiding knee joint proprioception, reproducing physiologic knee biomechanics, and maintaining femoral rollback . AlthougThe medial stabilized (MS-TKA) design was introduced in the early 1990s. Kinematic research demonstrated the medial compartment of the natural knee functioned like a ball-and-socket joint with the lateral femoral condyle translated in an anteroposterior direction and rotating around the medial compartment in flexion . MS-TKA Although the benefits of CR-TKA and MS-TKA have been reported in literature, the use of a MS knee design with a CR insert to preserve the PCL has not been reported. The combination should provide inherent stability and improved kinematics from the CR insert while the MS-TKA provides AP stability through the ball-and-socket joint. Therefore, this study aims to report on the patient-reported outcome measures (PROMs) and clinical and radiological evaluation of a MS knee with a CR insert.www.medacta.com) [The study was approved by the institutional research ethics committee. Patients who were scheduled for a TKA and met the inclusion criteria were invited to participate in the study and provide informed consent to be part of the study. Between September 2018 and November 2020, 177 MS-TKA  were implanted with a GMK Sphere CR tibial insert . All TKAs were performed by the senior author (J.H.) using the unrestricted caliper verified kinematic alignment (KA) TKA using manual instruments as per a previously described technique  ,26. The PROMs were the primary outcome measure for this study. Patients completed the Oxford Knee Score (OKS) and the Knee Injury and Osteoarthritis Outcome Score (KOOS) preoperatively and postoperatively at the 6-month and 1-year follow-up. The Forgotten Joint Score (FJS) and Visual Analogue Score (VAS) for Satisfaction were completed only at the postoperative timepoints. The secondary outcome was the clinical and radiological assessments. The clinical evaluation was assessed by measuring active knee flexion and knee extension both preoperatively and postoperatively at the 6-month follow-up timepoint. Radiological assessment for alignment was measured by the hip-knee-ankle angle (HKA), mechanical medial proximal tibial angle (mMPTA), and mechanical lateral distal femoral angle (mLDFA) via an anteroposterior long-leg weight-bearing radiograph.To calculate the mMPTA, the joint line of the proximal tibia was drawn preoperatively across the flat aspect of the subchondral line of the 2 tibial plateau. Postoperatively, the proximal tibial line was drawn parallel to the undersurface of the tibial component. The medial angle formed by the intersection of the joint line and the tibial mechanical axis was the mMPTA. The varus alignment was classified as mMPTA less than 90°, and the valgus alignment as greater than 90° .For the mLDFA, the preoperative joint line of the distal femur was drawn across the flat aspect of the subchondral line of the femoral condyles. The postoperative distal femoral line was drawn parallel to the undersurface of the femoral component. The lateral angle formed by the intersection of the joint line and the mechanical axis of the femur was the mLDFA. The valgus alignment was classified as mLDFA less than 90°, and the varus alignment as greater than 90°.t-test.Data were analyzed using SPSS, version 11 . Statistical significance was set at <0.05. The mean scores for the PROMs and clinical and radiological data were calculated for all timepoints and analyzed for statistical significance using the independent P = .0165 and P = .002, respectively). Similarly, an improvement in all the scores were observed between the preoperative and 1-year follow-up timepoints. The mean difference in improvement at 1 year for the KOOS subscales were 41.9 Symptoms, 45.8 Pain, 41.0 ADL, 52.3 Sport, and 56.9 QOL. The mean difference in improvement for the OKS was 20.4. There was a statistically significant difference between the preoperative and the 1-year postoperative follow-up, for the KOOS Symptoms (P = .001), Pain (P = .025), ADL (P = .005), Sport (P = .001), and QOL (P = .001), as well as for the OKS (P = .003). There was an improvement in the FJS (14.5) and the VAS Satisfaction (0.206) between the 6-month and 1-year follow-up timepoints; however, these were not statistically significant.The patient outcomes were compared between the preoperative and postoperative timepoints and between both post-operative timepoints as shown in P = .001). There was a statistically significant difference in the mean active extension between the preoperative and both postoperative timepoints .The active flexion and extension angles were captured and recorded in P = .001).The HKA angle, mMPTA, and mLDFA were captured preoperatively and at the 6-month postoperative timepoint . The meaP = .438). The mean preoperative and postoperative mLDFA were 87.5° (81.0°-96.3°) and 87.1° (80.5°-98.6°), respectively. There was no statistically significant change in the mLDFA between preoperative and postoperative timepoints (P = .929).The mean preoperative mMPTA was 86.7° (80.7°-92.1°), where 90.6% of the patients had a varus alignment, 5.7% had a valgus alignment, and the remaining had a neutral alignment. The mean postoperative mMPTA was 86.2° (80.3°-91.4°). There was no statistically significant change in the mMPTA between preoperative and postoperative timepoints . Similarly, a fluoroscopy study conducted by Iwamoto et al. assessing the in vivo kinematics following a CR-TKA reported a mean ROM of 121° (±17.3°) . The meaThe patient-reported outcomes demonstrated that the MS-TKA with a CR insert facilitates symptom relief and improves overall function of the knee joint after surgery. An improvement was seen as early as 6 months after the surgery, with a statistically significant improvement in both the KOOS subscales of Sport and Symptoms. In addition, the difference seen in KOOS scores at both 6 months and 1 year met the Minimal Clinically Important Difference (MCID) as established by Kuo et al. of 22.3 for Pain, 10.8 Symptoms, 22.1 ADL, 17.5 Sports, and 12.5 for QOL . SimilarMany studies have compared the clinical outcomes of the 3 different knee designs, where studies had found the superior performance of MS-TKA designs when reporting on CR-TKA, PS-TKA, and MS-TKA . The impThe limitation of this study was that there was no comparator group to compare the outcomes of the GMK Sphere CR to a medial stabilized design undertaken by the senior surgeon. All TKA were also conducted by a single experienced surgeon with a limited follow-up of 1 year postoperatively. Due to the single-surgeon cohort, the patients are from a localized area; therefore, the study is not reflective of a generalized population. All clinical evaluations for the ROM and radiological evaluation were conducted by the senior author (J.H.) during consultations, which can be a potential source of reliability error with intraobserver and interobserver reproducibility. Forty-eight TKAs were lost to follow-up in this study, which may be a possible limitation, as 27% of TKAs were not reported on due to intraoperative decisions to use another insert type. As such, this may have introduced selection bias to the study cohort. Not all the patellae were resurfaced within the study cohort. An analysis comparing whether the resurfaced or non-resurfaced patellae would have an impact on the outcomes was not conducted. Therefore, this is another possible limitation to the study.In summary, we found the patient-reported outcomes demonstrated that the MS-TKA with a CR insert facilitates symptom relief and improves overall function of the knee joint after surgery. Both the patient and clinical outcomes were comparable to 1-year outcomes utilizing other MS-TKA designs and were superior to the 1-year follow-up outcomes following implantation of CR-TKA. Most notably, the KOOS Symptoms and Sports scores were higher for the MS-TKA with a CR insert than for an MS-TKA design. This agrees with the high mean flexion angle achieved at 1-year follow-up. These findings suggest that the MS design coupled with the CR insert is advantageous in facilitating improved functional outcomes and restoring the quality-of-life following knee surgery.